Currently microbiological testing in the clinical setting relies on getting a primary culture on plate media, which will then undergo various identification testing and then antibiotic testing. This process usually takes a full 24-48 hours for the primary culture to grow large enough, then another 8-16 hours for antibiotic testing once the primary colony is large enough. For diseases, such as bacterial meningitis, this delay can be fatal within 24 hours of hospitalization, thus this time lapse makes current processes inadequate. Also, due to strain differences, the biochemical tests based upon the plate media can identify the bacteria as the wrong organism. Also, Gram staining, the gold standard of bacterial identification, can be difficult to properly do and can cause false identification. A more rapid and specific test is needed.
This invention is particularly helpful in the veterinary world when caring for large populations of animals. Currently it is impractical for ranch owners to test their entire herd for disease carriers. Instead of finding these carriers and performing targeted antibiotic therapy to only a few infected animals, most farmers and ranchers will treat the entire herd with antibiotics. This leads to increased resistance in the bacterial population as well as an increase in costs to market for the rancher. The molecular problem is that until now, in order to target multiple organisms, an individual primer/probe set was required for each target desired. This led to increasing assay complexity, false positives and decreased sensitivity the more targets that are assayed for, which the present invention overcomes.
A difficulty in developing assays for most real-time PCR thermocyclers is that most only have the ability to analyze up to four or five different color wavelengths. This has made multiplex assays at the clinical level meaningless. Only being able to assay at most 10 out of the 15-25 major bacterial species for each diagnostic sample media on a real-time PCR thermocycler was not worth the trouble due to the added costs of still being required to test for the rest of the bacterial species using the current standard culture method. It is for this reason that most Real-Time PCR machines are used for very specific assays for single organisms.
Researchers have tried to address the need for multiplexed bacteria detection. Most methods require a small amount of a primary culture, and then through various means are able to analyze the culture: by growth and/or biochemical tests (e.g., VITEK, Biomirieux): by analyzing chemical composition in a modified mass spectrometer (e.g., MALDI-TOF): by using antibodies paired to surface antigens (e.g., ELISA); or various other proprietary molecular probe based assays and equipment. All of these approaches have limitations, such as that they are only small scale and cannot be high-throughput, but the foremost being the requirement of a primary culture.
As a result, there exists the need for assays and methods for multiplexed detection of bacterial species, which are high-throughput, and able to test at least 40 strains of bacteria in one combined real-time PCR and HRMA assay with a run time of less than 4 hours.